WO2019168990A1 - Probiotiques et compositions probiotiques pour réguler le poids corporel - Google Patents

Probiotiques et compositions probiotiques pour réguler le poids corporel Download PDF

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Publication number
WO2019168990A1
WO2019168990A1 PCT/US2019/019845 US2019019845W WO2019168990A1 WO 2019168990 A1 WO2019168990 A1 WO 2019168990A1 US 2019019845 W US2019019845 W US 2019019845W WO 2019168990 A1 WO2019168990 A1 WO 2019168990A1
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Prior art keywords
bacteria
probiotic
subject
probiotic bacteria
modified
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PCT/US2019/019845
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English (en)
Inventor
Abhinav Prakash Acharya
Steven R. Little
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University Of Pittsburgh - Of The Commonwealth System Of Higher Education
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Publication of WO2019168990A1 publication Critical patent/WO2019168990A1/fr
Priority to US17/005,217 priority Critical patent/US11638727B2/en
Priority to US18/122,835 priority patent/US20230210918A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/30—Dietetic or nutritional methods, e.g. for losing weight
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/21—Streptococcus, lactococcus
    • A23V2400/245—Salivarius
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics

Definitions

  • the present disclosure relates to probiotics and probiotic compositions having modified energy metabolisms (e.g., modifying, metabolizing, or storing energy molecules, e.g., lipids, before the energy molecules are absorbed by a host subject), and thus regulating the absorption of the energy molecules by the host subject.
  • the present disclosure also relates to methods of making such probiotics and probiotic compositions.
  • the present disclosure further relates to methods of regulating (e.g., maintaining or reducing) body weight in a subject (e.g., a subject having a healthy body mass index (BMI), a subject having an overweight BMI, a subject having an obese BMI), using the probiotics and probiotic compositions disclosed herein.
  • BMI healthy body mass index
  • the present disclosure relates to probiotics and probiotic compositions having modified energy metabolisms, e.g., modifying, metabolizing, or storing energy molecules (e.g., lipids) before the energy molecules are absorbed by a host subject.
  • modified energy metabolisms e.g., modifying, metabolizing, or storing energy molecules (e.g., lipids) before the energy molecules are absorbed by a host subject.
  • the probiotics and probiotic compositions disclosed herein can regulate the absorption of the energy molecules by the host subject, e.g. at the gastrointestinal tract e.g., intestines, e.g., small intestine.
  • the present disclosure further relates to methods of regulating (e.g., maintaining or reducing) body weight in a subject, or treating a subject suffering from overweight or obesity, using the probiotics and probiotic compositions disclosed herein.
  • the present disclosure also relates to methods of making such probiotics and probiotic compositions.
  • the present disclosure provides a method of making a probiotic composition for treating an overweight subject including obtaining a microbiota sample from a subject, isolating bacteria from the microbiota sample, developing probiotic bacteria having modified metabolism by subjecting the isolated bacteria to a stress-based directed evolution, compared to isolated bacteria not subject to the stress-based directed evolution; and including the probiotic bacteria having modified metabolism in the probiotic composition.
  • the present disclosure provides a method of making a probiotic composition for treating an overweight subject including obtaining a microbiota sample from a subject, isolating bacteria from the microbiota sample, developing probiotic bacteria that utilize lipids as a source of energy by subjecting the isolated bacteria to a stress-based directed evolution; and including the probiotic bacteria having lipid metabolism in the probiotic composition.
  • the present disclosure provides a method of making a probiotic composition for treating an overweight subject including obtaining a microbiota sample from a subject, isolating bacteria from the microbiota sample, subjecting the isolated bacteria to a stress-based directed evolution to develop probiotic bacteria having increased metabolism as compared to the isolated bacteria that are not subject to stress-based directed evolution; and including the probiotic bacteria having increased metabolism in the probiotic composition.
  • the present disclosure provides a method of making a probiotic composition
  • a method of making a probiotic composition comprising: (a) obtaining a microbiota sample from a subject; (b) isolating bacteria from the microbiota sample; (c) subjecting the isolated bacteria to a stress-based directed evolution to generate the probiotic bacteria, wherein the probiotic bacteria have a modified metabolism as compared to the isolated bacteria obtained in step (b) ; and (d) incorporating the probiotic bacteria to the probiotic composition.
  • the present disclosure provides a method of making a probiotic composition comprising: (a) obtaining a microbiota sample from the subject;
  • step (b) isolating bacteria from the microbiota sample; (c) selecting probiotic bacteria from the isolated bacteria, wherein the probiotic bacteria have a modified metabolism as compared to the isolated bacteria obtained in step (b); (d) growing the probiotic bacteria to obtain an effective amount of the probiotic bacteria; and (e) incorporating the effective amount of the probiotic bacteria to the probiotic composition.
  • a probiotic composition comprises an effective amount of the probiotic bacteria disclosed herein and an acceptable carrier.
  • the probiotic composition further comprises one or more anti-obesity agents.
  • the present disclosure provides a method for treating an overweight or an obese subject including obtaining a microbiota sample from the subject, isolating bacteria from the microbiota sample, developing probiotic bacteria having modified metabolism by subjecting the isolated bacteria to a stress-based directed evolution; and administering to the subject an effective amount of the probiotic bacteria having modified metabolism.
  • the probiotic bacteria having modified metabolism utilize lipids as a source of energy.
  • the present disclosure provides a method for treating an overweight or an obese subject, including obtaining a microbiota sample from the subject, isolating bacteria from the microbiota sample, selecting, from the isolated bacteria, the bacteria having modified metabolism, culturing the isolated bacteria having modified metabolism to obtain an effective amount of the bacteria, and administering to the subject an effective amount of the bacteria having modified metabolism.
  • the present disclosure provides a method of inhibiting or reducing a lipid absorption in a gastrointestinal tract of a subject comprising: (a) obtaining a microbiota sample; (b) isolating bacteria from the microbiota sample; (c) subjecting the isolated bacteria to a stress-based directed evolution to generate probiotic bacteria, wherein the probiotic bacteria have a modified lipid metabolism as compared to the isolated bacteria obtained from step (b); and (d) administering to the subject an effective amount of the probiotic bacteria, wherein the probiotic bacteria inhibit or reduce an amount of lipids that is available for absorption in the gastrointestinal tract of the subject.
  • the present disclosure provides a method for regulating body weight of a subject comprising: (a) obtaining probiotic bacteria, wherein the probiotic bacteria have a lipid metabolism; and (b) administering to the subject an effective amount of the probiotic bacteria.
  • the present disclosure provides a method of regulating body weight of a subject comprising: (a) obtaining probiotic bacteria, wherein the probiotic bacteria have a lipid metabolism; (b) subjecting the probiotic bacteria obtained in step (a) to a stress-based directed evolution to generate probiotic bacteria having an increased lipid metabolism as compared to the probiotic bacteria obtained in step (a); and (c) administering to the subject an effective amount of the probiotic bacteria having the increased lipid metabolism.
  • the microbiota sample is a saliva sample or a stool sample.
  • the modified metabolism is a modified lipid
  • the modified lipid metabolism is a modified fatty acid metabolism.
  • the probiotic bacteria have a modified lipase activity.
  • the probiotic bacteria utilize lipids as a source of energy. In certain embodiments, the probiotic bacteria reduce a lipid absorption by the subject.
  • the probiotic bacteria decrease the amount of lipids available for absorption by the subject. In certain embodiments, the probiotic bacteria survive in a culture media comprising at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% v/v lipids. In certain embodiments, the stress-based directed evolution comprises culturing the isolated bacteria in increasing lipid concentrations.
  • the present disclosure provides a method of regulating body weight of a subject, comprising: (a) obtaining a microbiota sample; (b) isolating bacteria from the microbiota sample; (c) subjecting the isolated bacteria to a stress-based directed evolution to generate probiotic bacteria, wherein the probiotic bacteria have a modified metabolism as compared to the isolated bacteria obtained in step (b); and (d)
  • the present disclosure provides a method of regulating body weight of a subject, comprising: (a) obtaining a microbiota sample; (b) isolating bacteria from the microbiota sample; (c) selecting probiotic bacteria from the isolated bacteria, wherein the probiotic bacteria have a modified metabolism as compared to the isolated bacteria obtained in step (b); (d) growing the probiotic bacteria to obtain an effective amount of the probiotic bacteria; and (e) administering to the subject the effective amount of the probiotic bacteria.
  • the method maintains the body weight of the subject. In certain embodiments, the method reduces the body weight of the subject.
  • the subject has a healthy BMI. In certain embodiments, the subject has an overweight BMI. In certain embodiments, the subject is obese or has an obese BMI.
  • the microbiota sample is a saliva sample or a stool sample. In certain embodiments, the microbiota sample is obtained from the subject.
  • the modified metabolism is a modified lipid metabolism. In certain embodiments, the modified lipid metabolism is a modified fatty acid metabolism. In certain embodiments, the probiotic bacteria have a modified lipase activity. In certain embodiments, the probiotic bacteria utilize lipids as a source of energy. In certain embodiments, the probiotic bacteria reduces a lipid absorption by the subject. In certain embodiments, the probiotic bacteria decrease the amount of lipids available for absorption by the subject.
  • the probiotic bacteria survive in a culture media comprising at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% v/v lipids.
  • the stress-based directed evolution comprises culturing the isolated bacteria or probiotic bacteria in increasing lipid concentrations.
  • the present disclosure provides a probiotic composition comprising probiotic bacteria, wherein the probiotic bacteria have a modified
  • the probiotic composition is for use in regulating a body weight of a subject. In certain embodiments, the probiotic composition is for use in maintaining or reducing the body weight of the subject. In certain embodiments, the subject has a healthy BMI, an overweight BMI, or an obese BMI. In certain
  • the probiotic composition is for use in treating an obese or overweight subject.
  • the probiotic composition comprises an effective amount of the probiotic bacteria.
  • the modified metabolism is a modified lipid metabolism. In certain embodiments, the modified lipid metabolism is a modified fatty acid metabolism. In certain embodiments, the probiotic bacteria have a modified lipase activity. In certain embodiments, the probiotic bacteria have an increased lipid metabolism as compared to isolated bacteria that are not subject to stress-based directed evolution, wherein the isolated bacteria are isolated from a microbiota sample from a subject. In certain embodiments, the probiotic bacteria have an increased fatty acid metabolism as compared to isolated bacteria that are not subject to stress-based directed evolution, wherein the isolated bacteria are isolated from a microbiota sample from a subject. In certain embodiments, the probiotic bacteria have an increased lipase activity as compared to isolated bacteria that are not subject to stress-based directed evolution, wherein the isolated bacteria are isolated from a microbiota sample from a subject.
  • the probiotic bacteria reduce a lipid absorption by the subject. In certain embodiments, the probiotic bacteria survive in a culture media comprising at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% v/v lipids.
  • the probiotic composition further comprises an acceptable carrier. In certain embodiments, the probiotic composition further comprises one or more anti-obesity agent.
  • the probiotic bacteria having an increased lipid metabolism as compared to isolated bacteria that are not subject to stress-based directed evolution and/or the probiotic bacteria that utilize lipids as a source of energy inhibit lipid absorption by the subject by actively reducing the amount of lipids available for absorption by the subject.
  • kits including an effective amount of a probiotic composition disclosed herein.
  • the kits further comprise one or more anti-obesity agents.
  • the kits further comprise one or more weight management agents. 4. BRIEF DESCRIPTION OF THE FIGURES
  • FIGURE 1 shows that obesity has been linked to different disorders, such as type 2 diabetes, cardiovascular diseases, hypertension, stroke and certain forms of cancer and shows that the gut microbiota can be a preventive target of obesity.
  • FIGURE 2 shows that the microbiota actively modulates the nutrients absorbed in the body and can lead to excess fat deposition through absorption of lipid droplets through the epithelial layer.
  • FIGURE 3 shows that the microbiota actively prevents colonization of new bacteria.
  • FIGURE 4 shows the strategy to obtain probiotics of the disclosed subject matter.
  • Probiotics are isolated from the subject.
  • These probiotics undergo stress-based directed evolution. Stress-based directed evolution steps of repeated stress, screening and test are performed to isolate probiotics that have increased lipase expression and lipid metabolism, compared to isolated bacteria that were not subject to stress-based directed evolution.
  • the probiotics can be administered back to the subject.
  • These probiotics can have a higher chance of colonizing the gastrointestinal (GI) tract.
  • GI gastrointestinal
  • FIGURE 5 shows that the lipase activity was increased in probiotics by providing lipid stress.
  • FIGURE 6 shows a study design for the isolation and stress-based directed evolution of probiotics in saliva.
  • FIGURES 7A-7B show that probiotics of the disclosed subject matter can colonize the GI tract of littermates from which they were isolated at higher levels than a different strain of mice.
  • A Probiotics of the disclosed subject matter isolated from C57BL/6J mice (B6) were transformed with RFP plasmid and orally gavaged in B6 littermates of isolates and in BALB/c mice.
  • B Histology demonstrates that the probiotics of the disclosed subject matter B6 mice colonized in higher numbers in duodenum, jejunum, and cecum in B6 mice as compared to BALB/c mice.
  • FIGURES 8A-8B show that oral gavage of probiotics of the disclosed subject matter in the presence of high-fat diet prevents weight gain and in conjunction with diet- change leads to weight loss in mice.
  • A Mice that were orally gavaged with probiotics of the disclosed subject matter gain lower weight overall as compared to DH5a gavaged mice.
  • B Mice that were orally gavaged with probiotics of the disclosed subject matter lose weight faster in the presence of diet change as compared to DH5a+regular chow condition.
  • FIGURE 9 shows the representative image of mice that were given either DH5a control bacteria or probiotics along with high fat diet, at the end of the study. It was observed that the control group’s coat was shinier, and the total body size was bigger than the modified probiotics groups, suggesting lower accumulation of lipids.
  • FIGURES 10A-10B show the mice that are on high-fat diet and are given modified lipid metabolism probiotics gain substantially lower amount of weight as compared to the mice that are given high-fat diet and saline.
  • FIGURES 11 A-l 1E show that the presently disclosed probiotics did not significantly change the metabolism or overall health of the mice in the first 2 days.
  • A Study design.
  • B The energy expenditure (EE) data normalized to lean mass was not significantly different in the first 2 days between the mice receiving the same type of chow (HFD or RD) (Average ⁇ stderror).
  • C The total movement of mice was significantly higher for Probiotics + HFD group for the first 24 hours, after which this significance was not observed (Average ⁇ stderror).
  • D The food uptake was not significantly different between the mice getting the same type of chow (Average ⁇ stderror).
  • FIGURE 12 shows that the presently disclosed probiotics prevented weight gain in mice by reducing the build-up of fat mass in the body.
  • FIGURES 13A-13B show that the presently disclosed probiotics directly prevented build-up of fatty acids and triglycerides in the blood.
  • A Free fatty acid levels were significantly decreased in Probiotics + HFD group as compared to control bacteria + HFD group, even post-96 hours of treatment.
  • B Probiotics prevented the up- regulation of triglyceride levels in serum for 3 hours in mice and kept these levels significantly lower than the control bacteria.
  • FIGURES 14A-14C show that the presently disclosed probiotics induced higher excretion of fat in the stool samples of mice.
  • A Study design.
  • B Probiotics induced significantly higher level of fat excretion in mice as compared to control bacteria.
  • C Probiotics induced the excretion of Oleic acid significantly higher in mice as compared to control bacteria.
  • FIGURE 15 shows that Streptococcus intermedius family of bacteria were found to be the most common bacteria with lipase activity in dog saliva.
  • FIGURE 16 shows the proposed mechanism of action.
  • FIGURE 17 shows that the presently disclosed probiotics or nonevolved bacteria cultured in lipid-rich media differentially incorporated lipids intracellularly (dark shade - no lipids; light shade - presence of lipids).
  • FIGURE 18 shows that a benign bacterium Streptococcus salivarius was found to be the most common bacteria with lipase activity in human saliva.
  • the intestinal microbiota which are different among individuals, 4 plays an important role in nutrient absorption. 5 Moreover, the microbiota may play a role in intestinal absorption and extra-intestinal metabolism of dietary fat (Figure 2). 14 Colonization of probiotics in the intestine microbiota is often a difficult task. Existing microbiota, along with the immune system, actively inhibits colonization of new bacteria via prevention of adhesion, carbon source and micronutrient limitation. 15 Colonization by new bacteria may also be inhibited via IgA antibodies of the immune system ( Figure 3). 16
  • the present disclosure relates to a probiotics approach modifies the intestinal microbiota composition, and thus modulates the absorption of energy molecules by the host, for example, lipid absorption by the host at the intestines.
  • the present disclosure relates to a novel personalized probiotics approach, by utilizing a host subject’s own healthy oral and/or intestinal microbiota, for effective probiotic colonization at the gastrointestinal tract.
  • the word“a” or“an” when used in conjunction with the term“comprising” in the claims and/or the specification can mean“one,” but it is also consistent with the meaning of“one or more,”“at least one,” and“one or more than one.” Still further, the terms“having,”“including,”“containing,” and“comprising” are interchangeable and one of skill in the art is cognizant that these terms are open ended terms.
  • “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. In certain embodiments,“about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. In certain embodiments, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
  • the term“bacteria” encompasses both prokaryotic organisms and archaea present in mammalian microbiota.
  • the terms“intestinal microbiota”,“gut flora”, and“gastrointestinal microbiota” are used interchangeably to refer to bacteria in the digestive tract.
  • the terms“saliva microbiota,”“saliva flora,”“mouth microbiota,” and“mouth flora” are used interchangeably to refer to bacteria found in the oral cavity.
  • the term“abundance” refers to the representation of a given phylum, order, family, or genera of microbe present in the digestive tract of a subject.
  • “metabolism” can refer, for example, to lipid metabolism, fatty acid oxidation, or lipogenesis,.
  • modified metabolism or“change in metabolism” are used interchangeably to refer to a change in the metabolism and/or a change in energy consumption of a probiotic bacterium.
  • the change can be a decrease or an increase in the metabolism or in one or more metabolic pathways of fat and/or lipids of the isolated microbiota of a microbial species, genus, family, strain, order, or class.
  • the term“probiotics” or“probiotic bacteria” refers living bacteria that can be administered to a subject, e.g., orally consumed by a subject.
  • the presently disclosed probiotic bacteria or probiotics reduce the absorption of dietary lipids at the gastrointestinal tract of a subject. In certain embodiments, the presently disclosed probiotic bacteria or probiotics have the beneficial effects of modulating (e.g., maintaining or reducing) the body weight of a subject. In certain embodiments, the presently disclosed probiotic bacteria or probiotics have the beneficial effects of treating an overweight or obese subject.
  • the term“microbiota or bacteria having modified metabolism” as used herein can refer to isolated microbiota or bacteria subject to stress- based directed evolution so that they develop modified metabolism.
  • “modified metabolism” can refer to utilization of a specific molecule or category of molecules as a source of energy.
  • “modified lipid metabolism” can refer to utilization of lipids as a source of energy.
  • sources of energy can comprise lipids and/or fatty acids.
  • microbiota or bacteria having modified metabolism can refer to isolated microbiota or bacteria that exhibit modified metabolism, for example probiotic species having lipid metabolism, as observed by expression of lipase and/or other enzymes involved in lipid metabolism.
  • Probiotics usually do not readily utilize lipid metabolism as energy source and usually have very low, if any, expression of lipases and/or other enzymes involved in lipid metabolism. Therefore, probiotics having increased lipid metabolism can be selected and optionally subject to the directed-based evolution process.
  • probiotics having modified metabolism decrease quantities of energy molecules in the gastrointestinal (GI) tract when administered to a subject, thus decreasing absorption by the subject.
  • probiotics having modified metabolism modify the energy molecules, where the modified energy molecules cannot be or are difficult to be absorbed and/or metabolized by the subject. Therefore, change of the metabolism of the probiotics can lead to modification of the content of GI tract and modulation of the amount of energy sources that can be absorbed by the subject.
  • stress-based directed evolution of probiotics can refer to an ex-vivo introduction of environmental stressors to the isolated bacteria to enrich the bacteria for the desired trait and encourage the bacteria to enhance their protein production that favors their survival in the presence of stressors. The bacteria are then tested for the desired trait. The steps of screening and stressing can be repeated until the isolated bacteria can survive and proliferate in the presence of stressors.
  • exemplary stressors can include, for example, presence of lipids and/or fatty acids.
  • a“culture” of bacteria can refer to an in vitro culture of at least one bacterium species. Such bacteria can be cultured with one or more activators or repressors. As used herein, the terms“activators” and“repressors” refer to agents that increase or decrease the number and/or activity and/or metabolism of one or more desired bacteria, respectively.
  • the term“probiotic composition” can refer to a composition containing at least one species, genus, family, strain, order, or class of probiotic bacteria (e.g., a single isolate or a combination of desired bacteria), and can also include any additional carriers, excipients, and/or therapeutic agents that can be administered to a mammal.
  • the probiotic composition comprises a buffering agent to allow the probiotic bacteria to survive in the acidic environment of the stomach, that is, the probiotic bacteria resist low pH and are able to survive passage through the stomach to colonize and grow in the intestinal milieu.
  • Buffering agents can include, for example, sodium bicarbonate, milk, yoghurt, infant formula, and other dairy products.
  • the probiotic composition is formulated as a food additive.
  • the probiotic composition includes other materials known in the art for inclusion in food additives, such as water or other aqueous solutions, starch, binders, thickeners, colorants, flavorants, odorants, acidulants (e.g., lactic acid or malic acid, among others), vitamins, or minerals, among others.
  • carrier can refer to a diluent, adjuvant, excipient, or vehicle with which probiotic bacteria can be administered.
  • Such carriers can be, for example, sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water or aqueous solution, saline solutions and aqueous dextrose and glycerol solutions can be employed as carriers, particularly for injectable solutions.
  • the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant.
  • a binder for compressed pills
  • a glidant for compressed pills
  • an encapsulating agent for a glidant
  • a flavorant for a flavorant
  • a colorant for a colorant.
  • suitable carriers are described, for example, in“Remington's Pharmaceutical Sciences” by E. W. Martin.
  • “Patient” or“subject” or“individual” as used interchangeably herein, refers to a human or non-human subject.
  • Non-limiting examples of non-human subjects include non-human mammals, primates, dogs, cats, mice, rats, guinea pigs, rabbits, pigs, fowl, horses, cows, goats, sheep, cetaceans, etc.
  • Treatment (and grammatical variations thereof such as“treat” or“treating”) of a medical condition can include one or more of:
  • Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishing any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state and remission or improved prognosis; and
  • inhibiting the medical condition e.g., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or sub-clinical symptom thereof; and/or relieving the medical condition, e.g., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms.
  • the terms“inhibiting,”“reducing” or“prevention,” or any variation of these terms, referred to herein, includes any measurable decrease or complete inhibition to achieve a desired result.
  • the benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.
  • Treatment includes partial or full resolution of symptoms associated with the medical condition to be treated.
  • “Medical condition” can refer to any weight-related condition, including obesity (Body Mass Index, BMI of about 30 or higher), overweight (BMI of about 25 to about 30), and associated medical conditions and/or conditions where obesity can be a factor including hyperlipidemia, cancer, type 2 diabetes, hypertension, stroke, osteoarthritis, coronary heart disease, sleep apnea and respiratory problems, depression, gallbladder disease. A BMI of about 18.5 to about 25 is considered healthy. In certain
  • treatment of a subject and/or a healthy subject comprises maintaining a weight of the subject and/or the healthy subject and/or maintaining a BMI of about 18.5 to about 25 for the subject and/or the healthy subject.
  • an obese subject has a BMI of about 30 or higher
  • an overweight subject has a BMI of about 25 to about 30
  • a healthy and/or normal subject has a BMI of about 18.5 to about 25.
  • A“therapeutically effective amount” or“effective amount” as used herein can refer to an amount of a bacterial composition (probiotic) that, when administered to a subject for treating a medical condition, is sufficient to affect such treatment.
  • the “therapeutically effective amount” can vary depending on the composition administered as well as the stage of the medical condition and its severity and the age, weight, physical condition and responsiveness of the subject to be treated.
  • the phrase “acceptable” can refer to compositions that are generally regarded as physiologically tolerable to a patient.
  • microbiome as used herein can refer to the totality of microbes and their genetic elements (genomes) from a defined environment.
  • a defined environment can, for example, be the intestine and/or the oral cavity of a human being.
  • microbiome can include all area-specific microbiota and their complete genetic elements.
  • the presently disclosed subject matter relates to probiotics having a modified metabolism and probiotic compositions comprising thereof.
  • the present disclosure also relates to methods of making and modifying the presently disclosed probiotics.
  • the probiotics actively modulate processing of energy sources and, therefore, absorption at intestines.
  • the probiotics comprise personalized probiotic bacteria, probiotic bacteria having modified metabolism, or bacteria having a modified metabolism that actively consume energy sources in the gastrointestinal tract of a subject and can decrease the levels of energy sources that are available for absorption in the gastrointestinal tract of a subject.
  • the modified metabolism is lipid or fat metabolism.
  • the modified metabolism is a modified lipid or fat metabolism.
  • the modified lipid metabolism is an increased lipid or fat metabolism.
  • the probiotics and probiotic compositions disclosed herein can be administered to a subject in combination with a diet change from a high-fat in diet to a low-fat diet.
  • the present disclosure provides a method of making probiotics or a probiotic composition comprising thereof for maintaining body weight in a subject or treating an overweight subject comprising obtaining a microbiota sample from the subject and isolating bacteria from the microbiota sample.
  • Samples can be obtained and preserved using conventional techniques known in the art. Samples to be tested using the methods described herein can include, but are not limited to, saliva, tooth swab, tooth scrapping, cheek swabs, throat swab, sputum, endogastric sample, feces, and tissue biopsies.
  • the microbiota sample is a saliva sample.
  • the microbiota sample is a stool sample.
  • bacteria are isolated from the multiple species of microbial flora (e.g. Fungi).
  • microorganisms e.g., bacteria
  • Techniques for the isolation and cultivation of microorganisms include those, for example, described in the Manual of Clinical Microbiology, 8th edition; American Society of Microbiology, Washington D.C., 2003.
  • Bacterial co- cultures can be cultured according to standard practices.
  • techniques for the isolation of the microorganisms can be performed via centrifugation.
  • a microbiota sample can comprise one or more bacteria species selected from Table 1.
  • a microbiota sample can comprise bacteria of one or more of phyla Actinobacteria, Bacteroidetes, Chlamydiae, Chloroflexi, Euryarchaeota, Firmicutes, Fusobacteria, Proteobacteria, Spirochaetes, SR1, Synergistetes, Teneri cutes, and TM7.
  • a microbiota sample can comprise bacteria of one or more of Rothia genus, Escherichia genus, and specific species, for example and not by way of limitation, R. nasimurium.
  • a microbiota sample can comprise bacteria obtained from commercial sources, for example, ATCC.
  • individual species of the bacteria is isolated using lipolytic agar plates.
  • bacteria isolated from the saliva are identified using MALDI-TOF or comparative sequencing of the 16S ribosomal RNA (rRNA) gene in bacteria.
  • the isolated bacteria can be subject to stress-based directed evolution to develop probiotic bacteria having modified metabolism as compared to isolated bacteria that are not subject to stress-based directed evolution. Stress-directed evolution comprises introducing environmental stressors to the bacteria culture to enrich the bacteria for the desired trait and encourage the bacteria to enhance their protein production that favors their survival in the presence of stressors.
  • the bacteria thus isolated can be cultured in a 5 to 100 mL broth of energy source.
  • energy source can be in the form of lipids, for example but not by way of limitation, soybean oil, tween 20, tween 80 or any other lipid molecule.
  • the lipid concentration can range from 1% to 100% v/v in the broth.
  • the broth is made using by adding lipids to sterile saline solution at 1% to 20% vol/vol.
  • Table 1 Exemplary bacteria commonly found on the surfaces of the human body.
  • the isolated bacteria can be subject to stress-based directed evolution to develop probiotic bacteria having modified metabolism, compared to isolated bacteria that are not subject to stress-based directed evolution.
  • the modified metabolism comprises modified lipid metabolism.
  • the modified lipid metabolism comprises increased lipase expression. In certain embodiments, the modified lipid metabolism comprises increased expression of enzymes involved in lipid metabolism.
  • the lipid metabolism refers to fatty acid oxidation mediated energy generation and/or lipogenesis (generation of fat).
  • the isolated bacteria are grown on cholesterol as the sole carbon source in order to select probiotic bacteria that are capable of lowering cholesterol when administered to a subject. .
  • the probiotic bacteria having modified lipid metabolism are developed by culturing the isolated bacteria in increasing levels of lipids.
  • the isolated bacteria are cultured in a culture media having at least about 1% v/v lipids (1% lipids in 99% culture medium), then in a culture media having at least about 2% v/v lipids then in a culture media having at least about 5% lipids, then in a culture media having at least about 10% lipids, then in a culture media having at least about 20% lipids, then in a culture media having at least about 40% lipids, and then in a culture media having at least about 100% v/v lipids.
  • the percentage of lipid content in the culture is increased by at least about 5% v/v. In certain embodiments, the percentage of lipid content in the culture is increased by at least about 0.01% increment from 0% to 100% v/v lipid in the culture medium.
  • isolated bacteria are cultured in the presence of at least about 0.01%, of at least about 0.05%, of at least about 0.1%, of at least about 0.2%, of at least about 0.4%, of at least about 0.6%, of at least about 0.8%, at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, and/or at least about 100% v/v lipids in the culture medium.
  • the survival and proliferation of the isolated bacteria in the increasing levels of lipids is evaluated.
  • metabolic activities of the probiotic bacteria that are subjected to stress-based directed evolution are evaluated.
  • Probiotic bacteria having modified lipid metabolism can be isolated.
  • the probiotic bacteria having modified lipid metabolism utilize lipids as a source of energy.
  • the probiotic bacteria having modified lipid metabolism can prevent lipid absorption by the subject by actively decreasing the amount of lipids available for absorption in the gastrointestinal tract of the subject.
  • the probiotic bacteria having modified lipid metabolism can exhibit modified lipolytic activity, modified fatty acid metabolism, and/or modified lipase activity. In certain embodiment the probiotic bacteria having modified lipid metabolism can survive in culture medium containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% v/v lipids.
  • the probiotic bacteria subject to stress-based directed evolution can exhibit an increased metabolism that is at least about 1%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 50%, or at least about 100% increase as compared to isolated bacteria that are not subject to stress- based directed evolution.
  • probiotic bacteria having modified lipase activity are selected from a group of bacteria isolated from a saliva microbiota sample.
  • these probiotic bacteria can be cultured in an environment of modified or increasing lipid concentrations to further increase their dependency on lipids for energy.
  • lipase activity is measured in the isolated bacteria. In certain embodiments, the lipase activity is required for having lipids as an energy source.
  • lipids include triglycerides, glycerol, phospholipases, tween 80, tween 20, soybean oil, vegetable oil, olive oil, peanut oil, butyric acid, lauric acid, myristic acid, palmitic acid, stearic acid, Propionic acid - Propanoic acid, Butyric acid - Butanoic acid, Valeric acid - Pentanoic acid, Caproic acid - Hexanoic acid, Enanthic acid
  • Pentadecanoic acid Palmitic acid - Hexadecanoic acid, Margaric acid - Heptadecanoic acid, Stearic acid - Octadecanoic acid, Nonadecylic acid - Nonadecanoic acid - Arachidic acid - Eicosanoic acid, Heneicosylic acid - Heneicosanoic acid, Behenic acid - Docosanoic acid, Tricosylic acid - Tricosanoic acid, Lignoceric acid - Tetracosanoic acid, Pentacosylic acid - Pentacosanoic acid, Cerotic acid - Hexacosanoic acid,
  • the steps of screening and stressing can be repeated until the probiotic bacteria derived from the bacteria isolated from the microbiota sample can survive and proliferate in a modified lipid environment.
  • the “modified lipid environment” refers to an environment that includes an increased lipid level or a modified composition of different lipids (e.g., saturated fats, unsaturated fats) as compared to an environment where the bacteria are isolated from.
  • the“modified lipid environment” refers to an environment that includes dye labeled or unlabeled lipids, 13 C labeled or unlabeled lipids, deuterated or un- deuterated lipids, lipids that cannot be metabolized, and/or lipids that form complex structures (e.g. lipidoids, liposomes, micelles etc.)
  • the isolated bacteria are tested for their lipolytic activity and bacteria having modified lipolytic activity are selected.
  • the selected bacteria are either used in a probiotic composition or are first subject to a stress-based directed evolution and then used in the probiotic composition.
  • the isolated bacteria are grown in conditions similar to a modified fat diet, for example, a western-style diet.
  • Probiotic bacteria selected from the cultures are conditioned to flourish in an unhealthy environment, where the unhealthy environment comprises a modified lipid content.
  • the probiotic bacteria provide a therapeutic benefit following administration to an overweight subject.
  • the term“healthy diet” refers to a diet that can reduce the risk of cardiovascular diseases, cancer, diabetes and other conditions linked to obesity.
  • the term“healthy environment refers to an environment that comprises similar lipid levels and/or compositions as a healthy diet.
  • the term“modified fat diet” refers to a diet comprising an increased lipid level or a modified composition of different lipids (e.g., saturated fats, unsaturated fats) as compared to a healthy diet.
  • modified lipid content refers to an increased lipid level or a modified composition of different lipids (e.g., saturated fats, unsaturated fats) as compared to the lipid level or lipid composition in a healthy environment.
  • the probiotic bacteria having modified metabolism can be conditioned such that the probiotic bacteria are optimized for administration to a particular environment, for example, the intestine, a mucosal surface, etc.). That is, in the manufacturing process of a probiotic culture, a combination, of microbes is cultured such that they flourish in the gastrointestinal tract of a subject. In certain embodiments, probiotic bacteria having modified metabolism are also cultured with microbes expected to be in the environment to be treated. Such in vitro conditioning prior to in vivo administration can generate a bacterial culture that is able to survive the milieu of a target site that is contributing to a medical condition.
  • activators and/or repressors can be added to the cultures to enhance or decrease the metabolic activity of one or more species of bacteria.
  • Activators and repressors are known in the art. Genetic manipulation of probiotics for enhanced lipid metabolism or inhibition of enzymes such as phospholipases or lipases using small molecules for repressing metabolism can be used.
  • a probiotic composition or culture comprising culture of Rothia nasimurium and Escherichia coli bacteria to treat obesity.
  • a probiotic composition or culture comprising microbes of genera such as, for example, Rothia and Escherichia for treatment of obesity.
  • a culture for conditioning as described above for treatment of obesity which can comprise one or more of the following bacteria: Rothia nasimurium and Escherichia coli.
  • the probiotic bacteria having modified metabolism are included in the probiotic composition.
  • a probiotic composition can comprise one or more of the bacteria species included in Table 1.
  • a genetic analysis of the isolated bacteria is performed, to identify lineage and the susceptibility of these bacteria to different antibiotics and to determine pathogenicity.
  • the probiotic bacteria are personalized probiotic bacteria, where the probiotic bacteria having modified metabolism are administered to the same subject where the probiotic bacteria are derived from. Therefore, the probiotic bacteria are personalized and have higher capability of colonizing the intestinal mucosa because the probiotic bacteria are derived from bacteria that were part of the gut environment, and thus the immune system recognizes these bacteria as part of the microbiome.
  • personalized probiotic bacteria can allow the use of lower therapeutic amounts due to higher protein expression and can simultaneously allow the patient to avoid any potential harmful side-effects associated with reintroduction of specific bacterial strains.
  • the development of personalized probiotic bacteria having modified metabolism such as bacteria isolated by a subject can allow the use of lower therapeutic amounts due to higher metabolic activity and can further allow the subject to avoid any potential harmful side-effects associated with reintroduction of specific bacterial strains.
  • probiotic compositions comprising probiotic bacteria having modified metabolism as disclosed herein.
  • Probiotic compositions are formulated relative to an administration route.
  • a probiotic composition of the presently disclosed subject matter comprises an effective amount of probiotic bacteria having modified metabolism as disclosed herein, combined with an acceptable carrier.
  • Acceptable carrier includes any carrier which does not interfere with the effectiveness of the biological activity of the active ingredients and/or that is not toxic to the subject receiving such ingredients.
  • acceptable carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents and sterile solutions.
  • Additional non-limiting examples of compatible carriers can include any suitable vehicle, delivery or dispensing means or material.
  • Such acceptable carriers can be formulated by conventional methods and can be administered to the subject at an effective amount.
  • the probiotic composition is formulated as a food additive.
  • the food additive disclosed herein further comprises other materials known in the art for inclusion in food additives, including, but not limited, water or other aqueous solutions, starch, binders, thickeners, colorants, flavorants, odorants, acidulants (e.g., lactic acid or malic acid, among others), vitamins, minerals, and combinations thereof.
  • the food additive comprises between about 10 3 and about 10 4 CFU probiotic bacteria per gram of the food additive, between about 10 4 and about 10 5 CFU probiotic bacteria per gram of the food additive, between about 10 5 and about 10 6 CFU probiotic bacteria per gram of the food additive, between about 10 6 and about 10 7 CFU probiotic bacteria per gram of the food additive.
  • the present disclosure also provides a fortified food comprising the probiotics or probiotic compositions disclosed herein.
  • the fortified food disclosed herein further comprises a base food.
  • the food additive can be incorporated to a base food to form the fortified food. Any base foods known in the art can be used with the present disclosure.
  • Non-limiting examples of base foods include kefir, yakult, miso, natto, tempeh, kimchee, sauerkraut, water, milk, fruit juices, vegetable juices, yogurt, carbonated soft drinks, non-carbonated soft drinks, coffee, tea, beer, wine, liquor, alcoholic mixed drinks, bread, cakes, cookies, crackers, extruded snacks, soups, frozen desserts, fried foods, pasta products, potato products, rice products, corn products, wheat products, dairy products, confectionaries, hard candies, nutritional bars, breakfast cereals, bread dough, bread dough mix, sauces, processed meats, and cheeses.
  • a probiotic composition comprising probiotic bacteria having modified metabolism can be accomplished by any method likely to introduce the bacteria into the desired location.
  • the probiotics can be administered to a subject, in the form of a food additive or a fortified food disclosed herein, by oral consumption.
  • the probiotic bacteria can be mixed with a carrier and (for easier delivery to the digestive tract) be applied to liquid or solid food, feed, or drinking water.
  • the carrier material should be non-toxic to the bacteria and the subject/patient.
  • the carrier contains an ingredient that promotes viability of the bacteria during storage.
  • the formulation can include added ingredients to improve palatability and improve shelf-life. If a reproducible and measured dose is desired, the bacteria can be administered by a rumen cannula.
  • the carrier comprises a diluent, adjuvant, excipient, or vehicle with which probiotic bacteria are administered.
  • the carrier can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • the carrier can be water or aqueous solution, saline solutions and aqueous dextrose and glycerol solutions.
  • the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable carriers for therapeutic use are well known in the art and are described, for example, in“Remington's Pharmaceutical Sciences” by E. W. Martin, and in“Remington: The Science and Practice of Pharmacy.” Lippincott Williams & Wilkins.
  • the choice of a carrier can be selected based on the intended route of
  • oral delivery can be used for delivery to the digestive tract.
  • oral formulations comprise additional mixtures, such as milk, yogurt, and infant formula.
  • the duration and frequency of administration can vary between overweight and obese subjects or even between different subjects.
  • solid dosages in the form of tablets are used for the delivery of the probiotic bacteria by mixing the probiotic bacteria having modified metabolism with one or more components selected from the group consisting of sodium alginate, calcium carbonate, glyceryl monooleate, triethyl citrate, acetylated
  • HPMCAS hypromellose acetate succinate
  • the probiotic bacteria or probiotic compositions disclosed herein can be administered parenterally.
  • the probiotic bacteria or probiotic compositions of the presently disclosed subject matter can be prepared for delivery as a solution, a tablet, or as a lyophilized culture. Where cultures are lyophilized, the preparation can be rehydrated in, for example, yogurt or water for administration.
  • the probiotic bacteria or probiotic compositions of the presently disclosed subject matter are formulated such that they can survive passage through the acidic environment of the stomach and such that they adjust quickly to the intestinal environment.
  • Such formulation allows the presently described probiotic bacteria and probiotic compositions to have an elongated half-life in the intestines.
  • the probiotics or probiotic compositions disclosed herein are administered to a subject who has a healthy BMI. In certain embodiments, the probiotics or probiotic compositions disclosed herein are administered to a subject who has an overweight BMI. In certain embodiments, the probiotics or probiotic
  • compositions disclosed herein are administered to a subject who have a diagnosed disease, e.g., obesity.
  • the probiotics or probiotic compositions are administered to the subject in the form of food additives or fortified foods disclosed herein.
  • Dosage of the probiotic bacteria or probiotic composition disclosed herein for the subject can vary depending upon the characteristics of the subject (e.g., age, sex, race, weight, height, BMI, body fat percentage, and/or medical history), frequency of administration, manner of
  • the initial dose can be larger, followed by smaller maintenance doses.
  • the dose can be administered as
  • a variety of doses are effective to achieve colonization of the gastrointestinal tract with the desired probiotic bacterial, for example and not by way of limitation, about 10 6 CFU, about 10 7 CFU, about 10 8 CFU, about 10 9 CFU, about 10 10 CFU, about 10 11 CFU, about 10 12 CFU, about 10 13 CFU, about 10 14 CFU, or about 10 14 CFU of probiotic bacteria can be administered in a single dose to a subject.
  • lower doses can also be effective, for example and not by way of limitation, about 10 4 and about 10 5 CFU of probiotic bacteria.
  • the probiotic bacteria are administered to a subject in a dosage of between about 10 6 and about 10 7 CFU, between about 10 7 and about 10 8 CFU, between about 10 8 and about 10 9 CFU, between about 10 9 and about 10 10 CFU, between about 10 10 and about 10 11 CFU, between about 10 11 and about 10 12 CFU, between about 10 12 and about 10 13 CFU, between about 10 13 and about 10 14 CFU, or between about 10 14 and about 10 15 CFU.
  • the probiotic bacteria are administered to a subject in a dosage of about 10 10 CFU of probiotics.
  • the probiotic bacteria are administered to a subject in a dosage of up to about 10 12 CFU.
  • the subject is a human. In certain
  • the subject is a domestic animal, e.g., a canine.
  • the optimal dosage when administered to a subject having a diagnosed disease (e.g., obesity) or a subject having an obese BMI, can be empirically determined by treating physicians based on the stage of disease and patient statistics (e.g., age, height, weight, etc.). In certain embodiments, when administered to a subject who has a healthy BMI, or an overweight BMI, the optimal dosage can be empirically determined by the subject or a dietitian based on the subject’s statistics, e.g., age, sex, race, height, weight, BMI, body fat percentage, and/or medical history.
  • a diagnosed disease e.g., obesity
  • patient statistics e.g., age, height, weight, etc.
  • the optimal dosage when administered to a subject who has a healthy BMI, or an overweight BMI, can be empirically determined by the subject or a dietitian based on the subject’s statistics, e.g., age, sex, race, height, weight, BMI, body fat percentage, and/or
  • a probiotic composition or probiotic bacteria disclosed herein can be delivered every 4, 12, 24, 36, 48, 60, or 72 hours.
  • the probiotic composition or the probiotic bacteria can be delivered with at least one second pharmaceutically active ingredient, where the second pharmaceutically active ingredient can be delivered simultaneously or sequentially (e.g., within a 4, 12, 24-hour or l-week period) with the probiotic composition or the probiotic bacteria.
  • the probiotic composition or the probiotic bacteria can be delivered with two, three, four, five, or six second pharmaceutically active ingredients.
  • the treatment can last for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 2 months, at least about 3 months, at least about 6 months, or at least about 1 year.
  • one or more preparations of different probiotic bacteria having modified metabolism can be administered simultaneously (including
  • probiotic bacteria can be prepared from bacteria isolated from microbiota and then grown in a culture using known techniques to develop a modified metabolism.
  • bacteria species for example and not by way of limitation, Rothia nasimurium and/or
  • Escherichia coli can be administered orally at, for example and not by way of limitation at 10 7 CFU.
  • the probiotic composition further comprises one or more anti-obesity agent selected from the group consisting of an agent, a therapy, and a pharmaceutically active ingredient that is capable of negatively affecting obesity or weight gain in a subject, for example, by altering one of the fundamental metabolic processes of the host subject’s body, as opposed to the probiotic bacteria that themselves have one or more modified fundamental metabolic processes.
  • one or more anti-obesity agent selected from the group consisting of an agent, a therapy, and a pharmaceutically active ingredient that is capable of negatively affecting obesity or weight gain in a subject, for example, by altering one of the fundamental metabolic processes of the host subject’s body, as opposed to the probiotic bacteria that themselves have one or more modified fundamental metabolic processes.
  • the second pharmaceutically active ingredient can be an anti-obesity agent.
  • anti-obesity pharmaceutical agents include catecholamine release agents, such as amphetamine, phentermineTM and related substituted amphetamines (e.g. bupropion), agents that increase the human body’s metabolism, agents that interfere with the human body’s ability to absorb specific nutrients in food, for example and not by way of limitation, orlistat ® (tetrahydrolipstatin), loscaserin, sibutramine, rimonabant, metforminTM ( N,N-dimethylbiguanide), exenatide, phentermine, as well as herbal and dietary supplements.
  • catecholamine release agents such as amphetamine, phentermineTM and related substituted amphetamines (e.g. bupropion)
  • agents that increase the human body’s metabolism agents that interfere with the human body’s ability to absorb specific nutrients in food
  • orlistat ® tetrahydrolipstatin
  • loscaserin sibut
  • the probiotic composition can be administered in combination with at least one anti-obesity agent.
  • “In combination with,” as used herein, means that the probiotic composition and the one or more anti-obesity agents are administered to a subject as part of a treatment regimen or plan. In certain embodiments, being used in combination does not require that the probiotic composition and the one or more anti-obesity agent are physically combined prior to administration or that they be administered over the same time frame.
  • the probiotic composition and the one or more anti-obesity agent can be administered concurrently to the subject being treated or can be administered at the same time or sequentially in any order or at different points in time.
  • the present disclosure provides probiotic compositions comprising probiotic bacteria having modified metabolism wherein the probiotic bacteria having modified metabolism are at a concentration of between about 1 weight % and about 100 weight % (%w/w) of the probiotic compositions. In certain embodiments, the probiotic bacteria having modified metabolism are at a concentration of between about 1 ppm and about 100,000 ppm of the probiotic compositions. In certain embodiments, the probiotic bacteria having modified metabolism are at a concentration of about 1 pM of the probiotic compositions.
  • the development of personalized probiotic bacteria having modified metabolism can allow the use of lower therapeutic amounts due to higher metabolic activity and can further allow the subject to avoid any potential harmful side-effects associated with
  • probiotic bacteria having modified metabolism are administered to a different subject.
  • probiotic bacteria having modified metabolism are administered to the same subject.
  • the bacteria having modified metabolism administered to the same subject have higher capability of colonizing the intestinal mucosa because of bacteria has already been a part of the gut environment, and immune system recognizes these bacteria as part of the microbiome.
  • the presently disclosed matter relates to methods for treating an overweight subject, an obese subject, a subject aiming to maintain their body weight, a subject having normal BMI, or a subject otherwise in need of such treatment.
  • the methods comprise administering one or more probiotic compositions comprising probiotic bacteria having modified metabolism, disclosed herein.
  • the methods disclosed herein comprise obtaining a microbiota sample from the subject, isolating bacteria from the microbiota sample, developing probiotic bacteria having modified metabolism by subjecting the isolated bacteria to a stress-based directed evolution; and administering to the subject an effective amount of the probiotic bacteria having modified metabolism.
  • the methods disclosed herein comprise obtaining a microbiota sample from the subject, isolating bacteria from the microbiota sample, selecting probiotic bacteria having modified metabolism from the isolated bacteria, culturing the probiotic bacteria to obtain an effective amount of the probiotic bacteria, and administering to the subject an effective amount of the probiotic bacteria having modified metabolism.
  • the methods disclosed herein comprise obtaining a microbiota sample from the subject, isolating bacteria from the microbiota sample, subjecting the isolated bacteria to a stress-based directed evolution to develop probiotic bacteria that have increased metabolism as compared to the isolated bacteria that are not subject to the stress-based directed evolution, and administering to the subject an effective amount of the probiotic bacteria having increased metabolism.
  • the methods disclosed herein comprise obtaining a microbiota sample from the subject, isolating bacteria from the microbiota sample, selecting probiotic bacteria having modified metabolism from the isolated bacteria, culturing the probiotic bacteria to obtain an effective amount of the probiotic bacteria, and administering to the subject the effective amount of the probiotic bacteria having modified metabolism.
  • the presently disclosed matter relates to methods of inhibiting or reducing the lipid absorption in the gastrointestinal tract of a subject comprising obtaining a microbiota sample from a subject, isolating bacteria from the microbiota sample, developing probiotic bacteria having modified metabolism by subjecting the isolated bacteria to stress-based directed evolution, and administering to the subject an effective amount of the probiotic bacteria having modified metabolism to inhibit or reduce the lipid content that is available for absorption in the gastrointestinal tract of the subject.
  • the microbiota sample is a saliva sample.
  • the microbiota sample is a stool sample.
  • the methods disclosed herein comprise administrating to the subject an effective amount of a probiotic composition.
  • the probiotic composition comprises probiotic bacteria having lipid metabolism.
  • the probiotic composition comprises probiotic bacteria having modified lipid metabolism.
  • the probiotic composition comprises probiotic bacteria having increased lipid metabolism, compared to bacteria not subject to stress-based directed evolution.
  • the probiotic composition comprises probiotic bacteria having fatty acid metabolism.
  • the probiotic composition comprises probiotic bacteria having modified fatty acid
  • the probiotic composition comprises probiotic bacteria having increased fatty acid metabolism, compared to bacteria not subjected to stress-based directed evolution.
  • the probiotic composition comprises probiotic bacteria having modified lipase activity or other enzymes involved in lipid metabolism.
  • the probiotic composition comprises probiotic bacteria having increased lipase expression, compared to bacteria not subject to stress-based directed evolution.
  • the probiotic bacteria having modified metabolism prevent lipid absorption by the subject by actively decreasing the amount of lipids available for absorption by the subject.
  • the probiotic bacteria having modified metabolism survive in culture medium having at least about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% v/v lipids.
  • the probiotic bacteria having modified metabolism prevent lipid absorption by the subject by actively decreasing the amount of lipids available for absorption by the subject.
  • administering to the subject an effective amount of the probiotic bacteria having modified metabolism results in inhibiting weight gain in the subject and/or reducing body weight in the subject.
  • administering to the subject an effective amount of the probiotic bacteria having modified metabolism results in maintaining a normal BMI of a subject.
  • the probiotic bacteria having modified metabolism are administered to the same subject from whom the probiotic bacteria are derived. In certain embodiments, the probiotic bacteria having modified metabolism are
  • kits comprising an effective amount of a probiotic composition disclosed herein.
  • provided herein is a method of treating an overweight, an obese subject, a subject aiming to maintain their body weight and/or a normal BMI, or a subject otherwise in need of such treatment by administering orally a probiotic composition disclosed herein, wherein the probiotic composition comprises probiotic bacteria having modified metabolism, thereby decreasing quantities of energy molecules in the gastrointestinal (GI) tract when administered to a subject, thus decreasing absorption by the subject.
  • GI gastrointestinal
  • a subject provides a swab sample from the mouth. This sample is processed to isolate bacteria.
  • the isolated bacteria are cultured in vitro and probiotic bacteria are selected from the isolated bacteria for the specific trait of having highly active lipid metabolism.
  • the probiotic bacteria that specifically utilize large levels of fatty acid oxidation are isolated, grown in vitro , and are developed into a formulation. This formulation is then given to the subject, to be taken daily or alternate days, so as to increase the number of probiotic bacteria that metabolize or store lipids in the gastrointestinal tract of the subject.
  • the probiotic bacteria having modified lipid metabolism decrease the amount of lipids available for absorption by the subject, thereby decreasing the amount of lipids absorbed systemically through the intestines, and thus prevent weight gain in the subject.
  • lipid metabolism refers to fatty acid oxidation mediated energy generation and/or lipogenesis.
  • provided herein are methods for treating an overweight or obese subject by administering one or more probiotic compositions disclosed herein comprising one or more probiotic bacteria species, strain, or genus having modified metabolism.
  • the treatment can include administration of at least one of probiotic bacteria species or genus included in Table 1 and bacteria species or genus disclosed herein.
  • provided herein are methods for treating an overweight, an obese subject, a subject aiming to maintain their body weight and/or a normal BMI, or a subject otherwise in need of such treatment by administering one or more probiotic compositions disclosed herein comprising one or more probiotic bacteria species, strain, or genus having modified metabolism in combination with at least one anti-obesity agent.
  • treatment of an overweight or obese subject can also include one or more conventional regimens including, for example, bariatric surgery.
  • provided herein are methods for treating various diseases associated with obesity, for example and not by way of limitation, type 2 diabetes, cardiovascular, hypertension, stroke and certain forms of cancer, by administering the probiotic compositions disclosed herein.
  • metabolic conditions such as, for example, a disorder of fatty acid oxidation, steroid metabolism (e.g., congenital adrenal hyperplasia), abnormal lipid metabolism, and metabolic depletion of molecules in the gastrointestinal tract, for example and not by way of limitation cholesterol and allergy causing molecules.
  • metabolic conditions such as, for example, a disorder of fatty acid oxidation, steroid metabolism (e.g., congenital adrenal hyperplasia), abnormal lipid metabolism, and metabolic depletion of molecules in the gastrointestinal tract, for example and not by way of limitation cholesterol and allergy causing molecules.
  • the probiotic compositions described herein can be administered in combination with other therapeutic agents or regimes.
  • the choice of therapeutic agents that can be co-administered with the bacterial compositions depends, in part, on the condition being treated.
  • the probiotic bacteria-based treatment regimen can be further supplemented by a dietary change.
  • the dietary change includes decreasing dietary fat and/or increasing fiber consumption.
  • the dietary change further includes switching from a modified fat diet to a low-fat diet.
  • the dietary change is a switch from a modified lipid diet to a low lipid diet.
  • the probiotic bacteria based treatment regimen can be further supplemented by exercise.
  • the presently disclosed subject matter provides a kit for administering a probiotic composition of the presently disclosed subject matter.
  • the kit comprises an effective amount of one or more probiotic bacteria species, strain, or genus subject to stress-based directed evolution or a probiotic composition as described above. In certain embodiments, the kit comprises a probiotic composition comprising an effective amount of one or more probiotic bacteria species, strain, or genus subject to stress-based directed evolution. In certain embodiments, the kit comprises an effective amount of probiotic bacteria having modified metabolism or a probiotic composition comprising probiotic bacteria having modified metabolism as disclosed herein.
  • the kit can further include one or more components such as instructions for use, devices and additional reagents, and components, such as tubes, containers and syringes for performing the methods disclosed above.
  • the kit can further include one or more agents, e.g ., anti-obesity agents, that can be administered in combination with a probiotic composition.
  • the kit can include instructions for use, a device for administering the probiotic composition to a subject, or a device for administering an additional agent or compound to a subject.
  • the instructions can include a description of the probiotic composition and, optionally, other components included in the kit, and methods for administration, including methods for determining the proper state of the subject, the proper dosage amount, the proper administration method of administering the probiotic composition, and/or the proper storage of the kit. Instructions can also include guidance for monitoring the subject over the duration of the treatment time.
  • the kit can include a device for administering the probiotic composition and/or one or more agents, e.g. , anti-obesity agents to a subject.
  • a device for administering the probiotic composition and/or one or more agents e.g. , anti-obesity agents to a subject.
  • Any of a variety of devices known in the art for administering medications and probiotic compositions can be included in the kits provided herein. Non-limiting examples of such devices include, a hypodermic needle, an intravenous needle, a catheter, a needle-less injection device, an inhaler and a liquid dispenser, such as an eyedropper.
  • a probiotic composition and/or one or more agents, e.g. , anti-obesity agents to a subject to be delivered systemically, for example, by intravenous injection can be included in a kit with a hypodermic needle and syringe.
  • the kit can comprise one or more containers containing a probiotic composition, disclosed herein.
  • the kit can comprise one or more containers that contain probiotic bacteria comprising at one or more bacteria species, strain, or genus subject to stress-based directed evolution and/or a probiotic composition comprising probiotic bacteria having modified metabolism or a portion thereof.
  • Example 1 Probiotics of The Disclosed Subject Matter Have Higher Lipase Activity as Compared to Freshly Isolated Probiotics
  • the stab can be obtained using a sterile pipette tip.
  • the only other source of energy besides lipids was through the growth medium provided through LB broth.
  • the lipase activity is tested using lipase detection kit and the ability of the bacteria to survive in 100% lipid environment is evaluated. These five bacteria are then optionally genetically identified using MALDI-TOF microbial or 16S ribosomal RNA (rRNA) genotyping.
  • rRNA ribosomal RNA
  • Probiotics from human saliva are isolated and stress-based directed evolution on the probiotics isolated from human saliva is performed. If multiple species of microbial flora (e.g. Fungi) are isolated, saliva is first centrifuged at 300xGs to remove fungi and then centrifuged at 2500xGs to isolate bacteria. If the lipase activity is not increasing or if the bacteria are not able to survive in 100% tween 80 solution in PBS, then the percentage of lipid is increased 10% at a time (ex. 10%, 20%, 30%) to acclimatize the bacteria to lipid environment. Furthermore, five more bacterial colonies are isolated from initial plate if the above method is not successful, and the stress-based directed evolution is performed on these new set of bacteria. This process continues until at least one bacteria is isolated which can survive in 100% lipid environment.
  • microbial flora e.g. Fungi
  • Example 2 Probiotics of the Disclosed Subject Matter Can Colonize the GI Tract of Littermates from Which They Were Isolated at Higher Levels Than A Different Strain of Mice.
  • probiotics were isolated, cultured and delivery through gavage to litter-mate and non-littermate mice. Specifically, probiotics were isolated from the mouth of the mice using a Q-tip. It is important not to mix mice (or isolated bacteria) from different cages because mice are coprophagic, and as a result, littermates from the same cage essentially have the same microbiome, which can be vastly different than littermates in a different cage. These probiotics bacteria were then grown on a spirit blue agar plate and a colony with lipase activity was isolated (Figure 7A).
  • probiotic bacteria were then transformed using a red-fluorescent-protein (RFP) plasmid as a reporter gene.
  • RFP red-fluorescent-protein
  • the probiotic bacteria were grown in LB broth and re-administered to the mice (C57BL/6J littermates and BALB/c mice) using oral gavage. After 24 hours post-gavage, stomach, duodenum, ileum, jejunum, and cecum of mice were isolated. These tissues were then stained for nucleus and mounted on slides. The images ( Figure 7B) were then analyzed using a fluorescent microscope. As shown in Figure 7B, the probiotic bacteria colonized in the same littermates from which the bacteria were isolated.
  • Example 3 Oral Gavage of Probiotics of The Disclosed Subject Matter in The Presence of High-Fat Diet Prevents Weight Gain, and in Conjunction with Diet- Change Led to Weight Loss in Mice.
  • a diet- induced-obesity (DIO) model of C57BL6/J mice on high-fat diet (HFD) - 60% of Kcal coming from fat
  • HFD high-fat diet
  • Probiotic bacteria were isolated and subject to stress- based directed evolution in accordance with the methods disclosed in Example 1.
  • FIG. 10A shows the change of weight over the period of the experiment.
  • Figure 10B is a representative image of mice that are provided with high-fat diet and treated with saline and provided with high-fat diet and treated with the probiotics.
  • the fur of mice that are treated with saline was“ruffled,” whereas the fur of the mice treated with probiotics was normal. Without being bound to any particular theory, this shows that the mice consuming smaller amounts of probiotics actually absorbed lesser amount of fat.
  • the size of the mice treated with saline was larger than the mice treated with probiotics.
  • Example 4 Develop A Solid Oral Dosage Formulation for Probiotics of The Disclosed Subject Matter and Test Its Efficacy Post-Formulation in Mice.
  • probiotics of the disclosed subject matter to reduce weight in DIO mouse model (used in Example 3) when HFD fed mice are orally gavaged with probiotics of the disclosed subject matter in pill format in the presence of diet change is evaluated.
  • bacteria are isolated from mice and stress-based directed evolution is performed on these bacteria as described in Example 2. These bacteria are then harvested by centrifugation at 4,000 g for 25 min at 4°C. The cell mass is then mixed with sterile 15% glycerin made in PBS in a ratio 1 :5 (1 mL of glycerin for 5 g of pelleted cells).
  • mice are fed HFD ad libitum for 2 months. These mice are then switched to regular chow and orally gavaged with pill containing 10 7 CFU of evolved probiotics (alternate day for 2 months) and the weight is observed for 2 months.
  • the probiotics of the disclosed subject matter delivered in the form of a pill conjunction with reduced fat diet induce a more rapid and robust weight loss that no probiotic control in mice.
  • the CFU of probiotics are increased to 10 8 , 10 9 , or 10 10 in the pills and the weight-loss experiments are performed again.
  • pills are orally gavaged to HFD fed mice for 1 month (instead of 2 months) in order to test the effect on over-weight mice, instead of obese mice. If the pills create an issue as far as delivery of probiotics of the disclosed subject matter to the lower GI tract, or causing discomfort in mice, solid dosages in the form of tablets are developed by mixing probiotics of the disclosed subject matter with sodium alginate, calcium carbonate, glyceryl monooleate, triethyl citrate, and acetylated monoglyceride, and hypromellose acetate succinate (HPMCAS). 7-9
  • the ability of the probiotics of the disclosed subject matter to prevent weight gain in C57BL/6J mice is determined by providing mice the pills and high-fat diet (60% Kcal of fat) and monitoring the weight gain over a period of 1 month.
  • a genetic analysis of the isolated bacteria is also performed (MALDI-TOF/16S ribosomal RNA (rRNA) genotyping), in order to identify lineage and the susceptibility of these bacteria to different antibiotics and to determine pathogenicity.
  • rRNA ribosomal RNA
  • Example 5 Presently Disclosed Probiotics Do Not Modulate Host Metabolism as Evaluated by Energy Expenditure (EE) Adjusted for Lean Mass, Total Movement, Food and Water Intake at Thermoneutrality.
  • EE Energy Expenditure
  • the probiotic bacteria administered to the mice were isolated and underwent stress-based directed evolution in accordance with the method disclosed in Example 3. The mice were then transferred to metabolic cage and were kept on HFD for 96 hours. Experiments were performed at a constant 30 °C (thermoneutrality for mice) for the entire 96 hours.
  • Thermoneutrality was chosen for this experiment to ensure that the EE differences between the groups that were observed was mainly due to the probiotics administration.
  • mice were significantly lower in Probiotics + HFD vs. DH5a + HFD. Moreover, this difference accounted for 2 to 4 g (or 10% to 20% of the body weight), which has significant implications for preventing weight gain in larger animal models and humans.
  • Example 5 To determine if the change in the weight observed in Example 5 was due to decrease in fat or lean mass or both, after the metabolic cage experiment performed in Example 5, Echo-MRI DXA studies were performed. It was observed that there were no significant differences in fat mass between Probiotics + HFD, Probiotics + RD, and DH5a + RD; and these three were significantly lower than DH5a+HFD (Figure 12). These results suggest that the difference in weight gain was due to fat mass, which supports that the probiotics are preventing the transport of fat through the intestines and deposition in the body.
  • Example 7 Triglyceride Levels After Fat Gavage, And Fatty Acid Content was Significantly Lowered in Serum by The Probiotics as Compared to Control Bacteria.
  • mice were bled and euthanized. Serum was separated from blood and commercial assay kits for determining total fatty acid content was utilized. It was observed that the total fatty acid content was significantly higher in DH5a + HFD group as compared to all the other groups ( Figure 13 A). These data suggest that the probiotics actively prevented fatty acid build up in the blood, even though these mice were fed HFD.
  • mice were orally gavaged with soybean oil with either the probiotics or DH5a. The mice were then bled every hour, serum was separated from the blood and the amount of triglyceride in the serum was determined using a commercial assay kit. It was observed that the probiotics were able to prevent the increase in triglycerides in the serum as compared to control bacteria ( Figure 13B). These results support that the probiotics can prevent the transport of lipids through the intestines.
  • Example 8 The Measurement of Intestinal Fat Absorption Shows That the Presently Disclosed Probiotics Store/Consume Lipids.
  • mice were able to remove fat consumed through chow, HFD containing non-absorbable behenic acid (standard) was provided to mice along with alternate day of the probiotics or DH5a oral gavage (Figure 14A).
  • the probiotics administered to the mice were isolated and underwent stress-based directed evolution in accordance with the method disclosed in Example 3.
  • the stool samples from mice were collected and analyzed using mass-spectroscopy to determine the type and amount of lipids in the stool. It was observed that the mice receiving the probiotics excreted significantly higher (1.6 fold higher - Figure 9B) fat in the stool.
  • oleic acid major component of the fat mass in adipose tissue37,38 was determined to be excreted in significantly higher quantities in the probiotics group as compared to DH5a group (Figure 9C).
  • the presently disclosed probiotics are highly efficient at preventing weight gain in mice even though mice consume HFD, and also that weight loss is more efficient when the probiotics are combined with diet change. This change in weight can be attributed to the evolved probiotic bacteria storing/consuming the lipids inside the bacteria ( Figure 16 - proposed mechanism of action). Lastly, the probiotics do not change the metabolism of the host or lead to abnormal food and water intake and movement, supporting that lipids are being stored or metabolized by the evolved, personalized probiotic as opposed to being absorbed in the host.
  • Example 9 The Presently Disclosed Probiotics Modulated the Lipids in The
  • lipid-rich medium consisting of 40% LB broth (essential for obtaining a good yield of bacteria), 40% soybean oil (as the lipid source), and 10% tween 20 (as an emulsifier to ensure mixing of the oil and LB broth medium).
  • Controls included bacteria that did not undergo stress- based directed evolution cultured in lipid-rich medium, and lipid-rich medium by itself. After culturing for 16 hours at 37 °C at 200 rpm, supernatant and bacteria were separated.
  • Lipid extraction and methylation was performed to isolate the lipids, 44 and GC-MS and NIST library were utilized to identify the lipids.
  • the analysis suggested some of the lipids (specially for early elution time) were decreased in the supernatant in the presence of the probiotics. Moreover, new lipids were observed (later elution time) in probiotics’ supernatant (data not shown).
  • higher number of lipids were found in the probiotic bacteria as compared to the control bacteria ( Figure 17 - analysis, dark gray - no lipids, light gray - presence of lipids).
  • Example 10 Probiotics Can Be Isolated from Saliva of Dogs and Identified Using 16s rDNA
  • Dog saliva (one dog) was obtained and the presently disclosed probiotics from the dog saliva was generated. Specifically, dog saliva was obtained by swabbing a golden retriever male dog, and the Q-tip was stored at 4 °C until used. The saliva was then extracted, diluted (10 fold, 7 times) and spread on a Spirit Blue agar plate with 0.3% soybean oil as the source of oil. The colonies (a total of 4) that generated a halo on the plate were then picked, cultured in LB broth and l6s rDNA was utilized to identify the species of the bacteria. A representative Neighbor Joining Tree plot is shown in Figure 15.
  • Example 11 Probiotics Can Be Isolated from Saliva of Humans and Identified Using 16s rDNA
  • Human saliva was obtained, and the presently disclosed probiotics were generated from the saliva. Specifically, saliva was obtained by swabbing a male human, and the Q-tip was stored at 4°C until used. The saliva was then extracted, diluted (10 fold, 7 times) and spread on a Spirit Blue agar plate with 0.3% soybean oil as the source of oil. The colonies (a total of 4) that generated a halo on the plate were then picked, cultured in LB broth and l6s rDNA was utilized to identify the species of the bacteria. A representative Neighbor Joining Tree plot is shown in Figure 18. The most common species that survived and grew (largest colonies) was identified to be Streptococcus salivarius.
  • Streptococcus salivarius is one of the first colonizers of the human oral cavity and gut after birth and is known to prevent pathogen bacteria from colonizing.
  • Bacterial colonization factors control specificity and stability of the gut microbiota.
  • Phalipon A Cardona A, Kraehenbuhl JP, Edelman L, Sansonetti PJ, Corthesy B.
  • Secretory component a new role in secretory IgA-mediated immune exclusion in vivo. Immunity. Jul; 17(1): 107-15 (2002).

Abstract

La présente invention concerne des probiotiques et des compositions probiotiques ayant des métabolismes énergétiques modifiés (par exemple, la modification, la métabolisation ou le stockage de molécules énergétiques telles que des lipides, avant que les molécules énergétiques ne soient absorbées par un sujet hôte), et par conséquent modulant l'absorption des molécules énergétiques par le sujet hôte. La présente invention concerne également des procédés de production de tels probiotiques et compositions probiotiques. La présente invention concerne en outre des procédés de régulation (par exemple, maintien ou réduction) du poids corporel chez un sujet (par exemple, un sujet ayant un IMC sain, un sujet ayant un IMC de surpoids, un sujet ayant un IMC d'obésité), à l'aide des probiotiques et des compositions probiotiques selon l'invention.
PCT/US2019/019845 2018-02-27 2019-02-27 Probiotiques et compositions probiotiques pour réguler le poids corporel WO2019168990A1 (fr)

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* Cited by examiner, † Cited by third party
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WO2022051652A3 (fr) * 2020-09-04 2022-04-07 Arizona Board Of Regents On Behalf Of The University Of Arizona Bactéries orales génétiquement modifiées et leurs utilisations
US11369644B2 (en) 2018-04-10 2022-06-28 Siolta Therapeutics, Inc. Microbial consortia
US11406675B2 (en) 2019-10-07 2022-08-09 Siolta Therapeutics, Inc. Therapeutic pharmaceutical compositions

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US20120027736A1 (en) * 2005-07-26 2012-02-02 Nestec Ltd. Anti-obesity agent and anti-obesity food
US9113641B2 (en) * 2007-12-06 2015-08-25 Arla Foods Amba Probiotic bacteria and regulation of fat storage
WO2018022327A1 (fr) * 2016-07-28 2018-02-01 Bobban Subhadra Dispositifs, systèmes et procédés de production d'un microbiote commensal intestinal humanisé

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US20120027736A1 (en) * 2005-07-26 2012-02-02 Nestec Ltd. Anti-obesity agent and anti-obesity food
US9113641B2 (en) * 2007-12-06 2015-08-25 Arla Foods Amba Probiotic bacteria and regulation of fat storage
WO2018022327A1 (fr) * 2016-07-28 2018-02-01 Bobban Subhadra Dispositifs, systèmes et procédés de production d'un microbiote commensal intestinal humanisé

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Publication number Priority date Publication date Assignee Title
US11369644B2 (en) 2018-04-10 2022-06-28 Siolta Therapeutics, Inc. Microbial consortia
US11406675B2 (en) 2019-10-07 2022-08-09 Siolta Therapeutics, Inc. Therapeutic pharmaceutical compositions
WO2022051652A3 (fr) * 2020-09-04 2022-04-07 Arizona Board Of Regents On Behalf Of The University Of Arizona Bactéries orales génétiquement modifiées et leurs utilisations

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